JC's Lab Page
I have worked on a variety of toxins including ricin, shiga-like toxin 1 and diphtheria toxin. The main areas of my research have involved creating fusion proteins with regions of these different toxins including RA-IL2, RA-DTB and RA-SLTB. I am currently investigating abrin.
Abrin and ricin are found in the seeds of the indian licorise plant (Abrus precatorious) and the castor oil plant (Ricinus communis) respectively. The seeds are pictured below left with the Ricinus plant showing the spikey red seed pods to the right.
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My other Ricin Page
COOK JP, Savage PM, Lord JM, Roberts LM.
Biologically active interleukin 2-ricin A chain fusion proteins may require intracellular proteolytic cleavage to exhibit a cytotoxic effect.
Bioconjugate Chemistry. 1993 Nov-Dec;4(6): 440-7.
DNA fusions encoding chimeric proteins in which human interleukin 2 (IL2) was fused to the A subunit of the plant cytotoxin ricin (RA) have been expressed in Xenopus oocytes. The constructs contained N-terminal IL2 and C-terminal RA, or N-terminal RA and C-terminal IL2. In the expressed chimeric proteins, the IL2 and RA moieties were joined by a peptide sequence containing a proteolytic cleavage site. Two proteolytically-sensitive peptide sequences were utilized; a peptide that forms the trypsin-sensitive disulfide-bonded loop in diphtheria toxin (DT) or a synthetic peptide containing the factor Xa recognition site in a sequence flanked by two cysteine residues. In an in vitro cell free system the RA component was biologically active in all chimeric proteins produced since it specifically depurinated 28S ribosomal RNA. Proteolytic cleavage of the chimeras with either trypsin or factor Xa as appropriate separated the IL2 and RA moieties, but they did not remain covalently linked by a disulfide bond. Because of this, the cytotoxicity of protease-treated chimeras could not be assessed. Chimeras not pretreated with factor Xa but which contained the factor Xa target sequence were not cytotoxic to CTLL-2 cells. Rather, these molecules had a stimulatory effect that was ascribed to the IL2 moiety. In contrast, recombinant chimeric toxins containing the DT loop sequence were cytotoxic to CTLL-2 cells. Taken together the data suggest that RA-containing chimeras require intracellular proteolytic cleavage to release the RA moiety to render them cytotoxic to target cells.
An investigation of cleavable linkers in ricin A chain- interleukin 2 fusion proteins
CD40 ligand, Bcl-2, and Bcl-XL spare group I
Burkitt lymphoma cells from CD77-directed killing via Verotoxin-1 B chain but fail to protect against holotoxin.
Cell Death and Differentiation. (2000) 7: 785-794.
Owing to its lineage and differentiation stage-restricted expression, CD77 has been mooted as a therapeutic target in Burkitt lymphoma (BL). The recognition that the globotriaosyl moiety of this neutral glycosphingolipid is a receptor for Escherichia coli-derived Verotoxin-1 (Shiga-Like Toxin-1) offers a potential delivery system for the attack. Here we show that CD77-expressing Group I BL cells which are normally susceptible to activation-induced death on binding Verotoxin-1 B chain are protected in the presence of CD40 ligand. Ectopic expression of either bcl-2 or bcl-xL also afforded resistance to the actions of the B chain. In total contrast, neither of the survival genes nor a CD40 signal - even when acting in concert - protected against killing mediated by the holotoxin. These findings indicate that while therapeutic modalities for CD77-expressing B cell tumors (which include follicular lymphoma) based on the use of Verotoxin-1 B chain might be compromised by the activation of endogenous or exogenous survival pathways, those exploiting the holotoxin should be left unscathed
Ricin on the web
Some useful and some strange !
JC Cook 1999-2001